: A long term goal of the laboratory is to achieve an understanding of the Major Histocompatibility Complex (MHC) class I-restricted antigen processing pathway at a level that influences approaches toward cancer immunotherapy, transplantation, autoimmunity, and vaccine design. CD8+ cytotoxic T lymphocytes (TCD8+) play a critical role in limiting viral infections by lysing infected host cells before mature viral particles emerge and have been demonstrated to have powerful anti-tumor effects. As TCD8+ are major effectors of graft vs. host disease and may be involved in certain autoimmune diseases, there are also clear reasons for needing to suppress rather than amplify this population. TCD8+ are triggered by antigen-derived peptides (termed epitopes) held at the surface of the antigen-bearing cell by MHC class I molecules. In most cases the first step in the generation of peptide is degradation of antigen within the cytosol. Resultant fragments are then actively transported to the lumen of the endoplasmic reticulum where, perhaps following further modification, they bind to nascent MHC class I molecules. The heterotrimeric complexes are then conveyed to the cell surface where they can be contacted by T cells with appropriate receptor specificities. While a number of important discoveries have been made with respect to the processing pathway, a number remain to be discovered. What types of antigens feed most efficiently into the processing pathway? How influential is the rate at which a particular protein is synthesized and/or folded incorrectly. How do primary, secondary, and tertiary conformations of a protein influence antigen processing? How does the subcellular location of an antigen affect presentation? How do well-described degradation systems, such as the ubiquitindependent pathway, contribute to processing and are there cytosolic proteases other than the proteasome that participate in antigen processing? We hypothesize that properties of the antigen such as folding state, subcellular location, and primary sequence can strongly determine the efficiency with which any epitope is presented and that these properties carry different degrees of impact depending upon the protein. We will test these hypotheses by employing precise genetic manipulation of model antigens in combination with recombinant virus expression, quantitative in vitro and in vivo assays, and rigorous biochemistry. We anticipate that our work will be of value across a spectrum of fields, from fundamental cell biology to clinical medicine.